title: Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida.
abstract: Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5'-AMP, 3'-AMP, 2'-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.
authors.affiliation: Department of Chemistry, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Harkewal
authors.initials: H
authors.lastname: Singh
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Malinski
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Reilly
authors.forename: Michael T
authors.initials: MT
authors.lastname: Henzl
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D008958:Models, Molecular; D016979:Pasteurella multocida / Q000737:chemistry / Q000201:enzymology* / Q000276:immunology* / Q000302:isolation & purification; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D000135:Acid Phosphatase